HMGA1 induces FGF19 to drive pancreatic carcinogenesis and stroma formation

High mobility group A1 (HMGA1) chromatin regulators are upregulated in diverse tumors where they portend adverse outcomes, although how they function in cancer remains unclear. Pancreatic ductal adenocarcinomas (PDACs) are highly lethal tumors characterized by dense desmoplastic stroma composed predominantly of cancer-associated fibroblasts and fibrotic tissue. Here, we uncover an epigenetic program whereby HMGA1 upregulates FGF19 during tumor progression and stroma formation. HMGA1 deficiency disrupts oncogenic properties in vitro while impairing tumor inception and progression in KPC mice and subcutaneous or orthotopic models of PDAC. RNA sequencing revealed HMGA1 transcriptional networks governing proliferation and tumor-stroma interactions, including the FGF19 gene. HMGA1 directly induces FGF19 expression and increases its protein secretion by recruiting active histone marks (H3K4me3, H3K27Ac). Surprisingly, disrupting FGF19 via gene silencing or the FGFR4 inhibitor BLU9931 recapitulates most phenotypes observed with HMGA1 deficiency, decreasing tumor growth and formation of a desmoplastic stroma in mouse models of PDAC. In human PDAC, overexpression of HMGA1 and FGF19 defines a subset of tumors with extremely poor outcomes. Our results reveal what we believe is a new paradigm whereby HMGA1 and FGF19 drive tumor progression and stroma formation, thus illuminating FGF19 as a rational therapeutic target for a molecularly defined PDAC subtype.

control (0.5% carboxymethylcellulose) twice daily by oral gavage. Weekly ultrasounds were performed to monitor tumor growth.
Transwell inserts were coated with a thin layer of Matrigel for invasion (Corning). Before seeding into upper chambers, cells (~1.5x10 5 cells) were maintained in serum-free medium for 24 h and treated with 10 µM of Cytosine β-D-arabinofuranoside (AraC, Sigma) to block proliferation (7). Complete medium was added to the lower chambers as a chemoattractant; migration or invasion was assessed after 24 h by fixation (4% paraformaldehyde) and crystal violet stain (0.2%; Sigma) for enumeration.

RNA isolation and quantitative reverse transcriptase (RT) PCR analysis (qPCR):
Total RNA from cells was isolated using Trizol reagent (Invitrogen) and the Direct-zol RNA miniprep kit (Zymo Research) according to the manufacturer's protocol. RNA (500 ng) was used for generating cDNA using the High-capacity cDNA Reverse Transcription kit (Applied Biosystems) according to the manufacturer's protocol. Expression for genes of interest with primers (Supplemental Table 3) was detected in human and mouse samples by quantitative PCR (qPCR), and reactions were performed using Power SYBR Green Master Mix (Applied Biosystems) on QuantStudio 3 (Applied Biosystems). Relative expression was determined as described with HuPO as a loading control in human cells and Gapdh as a loading control in mouse samples (14).
Chromatin Immunoprecipitation: Cells (10 7 ) were washed with phosphate-buffered saline (PBS) and crosslinked with formaldehyde (1%) for 10 minutes at room temperature. Glycine (1.25 M) was added to quench the crosslinking reaction, and cells were subsequently washed twice with cold PBS. Cells were lysed in cell lysis buffer (20 mM Tris pH 8.0, 85 mM KCl, and 0.5% NP40) containing protease inhibitors (Millipore Sigma) and centrifuged at 430 g for 5 minutes at 4°C to separate cytoplasmic and nuclear fractions. Nuclear pellets were collected, lysed in nuclei lysis buffer (10 mM Tris-HCl pH 7.5, 1% NP40, 0.5% Na Deoxycholate, 0.1% SDS) containing protease inhibitors, transferred into a 1 ml milliTUBE (Covaris) and sheared using Covaris S220 (Covaris) sonicator. An aliquot of sheared chromatin (10%) was prepared as input. The sheared chromatin was immunoprecipitated by incubating with antibodies and Protein A/G Dynabeads (mixed 1:1, ThermoFisher Scientific) overnight while rotating at 4°C with the specified antibodies (Supplemental Table 4). Immunoprecipitates were washed with wash buffer (10 mM Tris-HCl pH 7.5, 1% NP40, 0.1% SDS) and eluted with ChIP elution buffer (50 mM NaHCO3 and 1% SDS). Immunoprecipitated DNA samples were treated with RNase A (ThermoFisher Scientific) and proteinase K (ThermoFisher Scientific) and subsequently purified (QIAquick PCR Purification Kit; Qiagen). ChIP DNA was subjected to qPCR analysis using Power SYBR Green Master Mix (Applied Biosystems). MatInspector in silico transcription factor binding site prediction algorithm (15) was used to design primers for ChIP-qPCR analysis. (Primers are listed in Supplemental Table 3). ChIP-qPCR results are represented as a percentage of input based on mean quantity derived from a standard curve (4,14).
Luciferase constructs and reporter assay: Luciferase reporter plasmids expressing FGF19 promoter sequences containing predicted HMGA1 binding sites (-1144, -1046, -816, and -756 base pairs from the transcription start site) determined by MatInspector (15) were generated by PCR and cloned into pLightswitch_prom Renella luciferase plasmid (Switchgear Genomics). Sequences of the constructs were confirmed by Sanger sequencing. E3LZ10.7 cells with or without HMGA1 silenced were transfected using Lipofectamine 3000 (Thermo Fisher Scientific) and a mixture of Renella luciferase vector (320 ng) and control PGK-Firefly luciferase vector (100 ng; Promega). At 48 h following transfection, cells were lysed using cell lysis buffer (Promega), and luciferase activity was measured. Renella luciferase activity was measured using the LightSwitch assay reagent (SwitchGear Genomics), while firefly luciferase activity was measured using the Luciferase assay reagent (Promega). Expression of the reporter gene was calculated as fold induction over the full-length promoter construct.
In vivo limiting dilution assay: Cells were injected into both flanks of the nude mice in a 1:1 mixture of PBS:Matrigel (Corning); AsPC-1 studies were performed without Matrigel due to supply chain disruptions during the pandemic. Mice were followed biweekly for tumor formation and underwent sacrifice and necropsy once tumor diameter reached 10 mm in mice injected with control cells. Tumor initiator frequency was calculated using Extreme Limiting Dilution Analysis (16) where P value was determined by the chi-square test. Tumor volume was calculated as described (17) with volume = 0.5 x l x w 2 (l: length; w: width of tumor).

Immunohistochemistry and immunofluorescence: Hematoxylin & eosin (H&E) and immunohistochemistry (IHC)
of xenograft sections and KPC pancreas were performed after formalin fixation and paraffin embedding as described previously (4,18,19). Slides were rehydrated in xylene and ethanol series after baking at 70°C for 1 h. Antigen retrieval was performed using preheated Target Retrieval Solution (Dako, S1699) for 20 minutes at 68ºC followed by 20 minutes at 99ºC. Slides were allowed to cool for 30 minutes followed by Tris Buffered Saline (TBS) with 0.5% Tween (Sigma Aldrich, TBST) washes. Slides were then blocked in 3% H2O2 for 30 minutes followed by Dako dual endogenous enzyme block (Dako, S200389-2) for 30 minutes and washed with TBS with 0.5% Tween and incubated with 3% Bovine Serum Albumin solution for 30 minutes. Tissues were incubated overnight at 4°C with primary antibodies (Supplemental Table 4) diluted in Dako antibody diluent (Dako, S080983-2). Tissues were subsequently washed in TBS with 0.05% Tween 20 and incubated with secondary antibodies (Supplemental Table 4 DAPI staining at 20x magnification and enumerated total 1154 ± 549 CAFs (mean + SD CAFs/field) based on staining for podoplanin (PDPN, a "panCAF" marker). CAF composition was calculated as the percent of total PDPN+ cells that co-stain for another CAF marker, including and: 1) α-smooth muscle actin (α-SMA), 2) CD74, a transmembrane molecule involved in the formation and transport of major histocompatibility (MCH) class II peptides, and, 3) IL6, an inflammatory cytokine. A total of 10 fields were selected in tumors from 1-2 mice for each condition.
Immunoblotting: Total proteins were isolated from PDAC cells using RIPA buffer (Millipore Sigma) containing Halt protease and phosphatase inhibitor cocktail (ThermoFisher Scientific). Lysates (20 or 30 ug; with the same quantity loaded per lane per run) were separated on a gradient (4-12%) bis-tris gel (ThermoFisher Scientific) via SDS-polyacrylamide gel electrophoresis, transferred onto a polyvinylidene fluoride membrane (ThermoFisher Scientific), and analyzed using various primary antibodies (Supplemental Table 4). Beta-Actin (β-Actin) was used as a loading control in all Western blots. Most bands (HMGA1, β-Actin) on western blots were visualized with horse radish peroxidase (HRP) using a commercial imager (ChemiDoc XRS+; Bio-Rad) for the following: bands were difficult to detect with HRP and the prior imager (ChemiDoc XRS+; Bio-Rad), we used a more sensitive, near infra-red fluorescence detection system (Odyssey CLx Imager; LI-COR Biosciences) to compare FGF19 and β-Actin in E3LZ10.7 and MIA PaCa-2 cells (Figures 3E, 4B). FGF19 (Santa Cruz), HMGA1 (Abcam), and b-Actin (Cell Signaling Technologies) were detected on the same blots shown in each figure section. HMGA1 and b-Actin were detected at 800 nm wavelength, and FGF19 was detected at 700 nm on the same blot in each figure section (Odyssey CLx Imager; LI-COR Biosciences). In blots from MIA PaCa-2 cells, the blot was stripped for 30 minutes at 37 ºC in stripping buffer (ThermoFisher Scientific). The blot was washed 3 times with TBS with 0.1% tween and re-probed with FGF19 antibody (Santa Cruz). FGF19 was then detected at 700 nm wavelength (Odyssey CLx Imager; Li-COR Biosciences).
Enzyme-linked immunoassay (ELISA) and cytokine array: For cytokine secretion analyses, cells were cultured in serum-free medium without phenol red for 24 h, after which medium was collected and centrifuged at 2,500 g for 10 minutes, and the resulting supernatant was concentrated onto filters (Amicon Ultra centrifugal filters -10 k; Millipore Sigma). The concentration of secreted proteins was measured using a commercial protein assay (Coomassie Plus protein assay reagent; ThermoFisher Scientific). Total secreted proteins (200 ng for E3LZ10.7 and AsPC-1 cells, and 250 µg for MIA PaCa-2 cells) were used to quantitatively assess FGF19 by ELISA (RayBiotech) according to the manufacturer's protocol. Total secreted protein (750 µg) was used to quantify cytokines secreted in E3LZ10.7 cells using the Proteome Profiler Human XL Cytokine Array Kit (RnD Systems) according to manufacturers' protocol. Statistical analysis: When comparing 2 groups, statistical significance was determined using a two-tailed student's t-test when normally distributed (ascertained by Ryan-Joyner and D'Agostino-Pearson tests). If not normal, the Mann-Whitney test was used. When comparing more than 2 groups, statistical significance was determined using a one-way ANOVA with Dunnett's or Turkey's multiple comparisons (Prism 9, GraphPad Software). Survival analyses were performed under the assumption of Cox proportional hazards and evaluated by log-rank test. Tumor initiator cell frequency was compared by chi-square distributions. P<0.05 was considered significant. (F) HMGA1 expression in cells with HMGA1 silencing before injection and from tumors that formed following injection (n=3 tumors from E3LZ10.7 cells with HMGA1 silencing). (G) HMGA1 expression in cells with HMGA1 silencing before injection and from tumors that formed following injection (n=7 tumors from AsPC-1 cells with HMGA1 silencing). (H) Xenograft tumorigenicity at limiting dilutions in E3LZ10.7 ± HMGA1 silencing. Tumor initiator cell frequency assessed by extreme limiting dilution analysis (ELDA; n=10/condition). Data show 95% confidence interval around tumor initiator cell frequency. P values determined by chi-square test. (I) Xenograft tumorigenicity at limiting dilutions in AsPC-1 ± HMGA1 silencing. Tumor initiator cell frequency assessed by ELDA (n=10/condition). Data show 95% confidence interval around tumor initiator cell frequency. P values determined by chi-square test. Data shown as mean ± SD. P values determined by one-way ANOVA with Dunnett's multiple comparisons (A, B, D, E) or Turkey's multiple comparisons (F, G). *P < 0.05, **P < 0.01, ****P < 0.0001.